Amide-to-ester substitution as a stable alternative to N-methylation for increasing membrane permeability in cyclic peptides

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.


Supplementary Notes
Chemicals used in this study were purchased from commercial suppliers as received. Preparative HPLC was performed on a Prominence HPLC system (Shimadzu) with a 5C18-MS-II column (Nacalai tesque, 10 mm I.D.×150 mm, 34355-91).

Synthesis of (S)-3-[4-tert-butoxyphenyl]-2-hydroxy-propionic acid (HO-Tyr(tBu)-OH)
The compound was synthesized according to the previous report 1 with minor modifications. O-tert-butyl-L-tyrosine, or H2N-Tyr(tBu)-OH (6.0 mmol, 1.4 g) was dissolved in 8:2 water:acetic acid (v/v, 60 mL) and cooled on ice. 6.0 mL (12 mmol) of 2 M sodium nitrite aqueous solution was added to the H2N-Tyr(tBu)-OH solution dropwise on ice. After 15 minutes, the reaction solution was allowed to warm to room temperature and stirred overnight. The reaction was monitored using thin-layer chromatography (TLC). After finishing the reaction, the reaction was quenched with 0.42 mL (5.0 mmol) of 40% methylamine aqueous solution. The quenched solution was acidified up to pH 3 with 1 M aqueous hydrogen chloride and extracted with ethyl acetate (3 × 50 mL). The organic phase was dried using sodium sulfate, filtered, and evaporated under vacuum. The product was further purified by column chromatography on silica gel using 90:9:1 chloroform:methanol:acetic acid to give the objective compound as white solid. Yield 0.52 g (2.2 mmol, 40%

Synthesis of Dimer peptides (P1-3)
The peptide was manually synthesized on Sieber amide resin (0.55 mmol/g). Resin (20 mg, 11 µmol) was swelled with N,N-dimethylformamide (DMF) in a 6 mL fritted syringe with continuous shaking. Fmoc deprotection was performed by incubating the resin with 20% piperidine/DMF for 2 min with continuous shaking. After washing the resin with DMF, the residual Fmoc group was removed by incubating the resin with 20% piperidine/DMF for 8 min with continuous shaking, and the resin was washed with DMF three times. Fmoc-protected amino acids (Fmoc-Leu-OH or Fmoc-Phe-OH) (4 equivalent), HATU (4 equivalent), and N,N-diisopropylethylamine (DIPEA) (8 equivalent) were dissolved in 440 µL of DMF, and the solution was added to the resin. The resin was incubated for 1 h at room temperature with continuous shaking.
After the reaction, the resin was washed with DMF three times. Fmoc group was removed in the same way as described above, and the N-terminal residue was coupled for 4 h using Fmoc-protected amino acids (Fmoc-Leu-OH or Fmoc-Phe-OH) (4 equivalent), HATU (4 equivalent), and DIPEA (8 equivalent) in 440 µL of DMF. After the reaction, the resin was washed with DMF three times. After removal of the Fmoc group, 0.5 M acetic anhydride and 1 M DIPEA in 1 mL of DMF were added to the resin. The resin was incubated for 1 h at room temperature with continuous shaking. After removal of the reaction solution, the resin was washed with DMF and dichloromethane (DCM) three times each. Peptides were cleaved from the resin by shaking the resin with 95% trifluoroacetic acid (TFA)/2.5% triisopropylsilane (TIPS)/2.5% water for 2 h twice. The solution was evaporated. 10% acetonitrile/water was added to the peptides, and the peptides were purified by HPLC. Purified peptides were dissolved in N,N-dimethylsulfoxide (DMSO), yielding 10 mM peptides solution as DMSO stock. The concentration was determined based on the weight of the peptide. Purified peptides were analyzed by ESI-MS and UPLC.

Dimer depsipeptides (D1-3)
The peptide was manually synthesized on Sieber amide resin (0.46 mmol/g). Resin (40 mg, 18.4 µmol) was swelled with DMF in a 6 mL fritted syringe with continuous shaking. Fmoc deprotection was performed by shaking the resin with 20% piperidine/DMF for 3 min. After washing the resin with DMF, the residual Fmoc group was removed by shaking the resin with 20% piperidine/DMF for 12 min, and the resin was washed with DMF three times. α-Hydroxy acids (L-Leucic acid or L-3-Phenyllactic acid) (8 equivalent), HATU (8 equivalent), and DIPEA (16 equivalent) were dissolved in 736 µL of DMF, and the solution was added to the resin. The resin was shaken for 3 h at room temperature. After the reaction, the resin was washed with DMF and tetrahydrofuran (THF) three times each. The coupling of Fmoc-protected amino acids to α-hydroxy acid immobilized on the resin was performed according to a previous report. 2 Fmoc-amino acids (Fmoc-Leu-OH or Fmoc-Phe-OH) (8 equivalent), diisopropylcarbodiimide (DIC) (8 equivalent), and N,N-dimethylaminopyridine (DMAP) (0.2 equivalent) were dissolved in 736 µL of THF, and the solution was added to the resin. The resin was shaken for 2.5 h at room temperature. After the reaction, the resin was washed with THF and DMF three times each. Fmoc deprotection was performed by shaking the resin with 20% piperidine/DMF for 2 min. After washing the resin with DMF, the residual Fmoc group was removed by shaking the resin with 20% piperidine/DMF for 8 min, and the resin was washed with DMF three times. Acetic acid (4 equivalent), Oxyma (4 equivalent), and DIC (4 equivalent) were dissolved in 368 µL of DMF and preincubated for 10 min, and the solution was added to the resin. The resin was incubated for 1.5 h at room temperature with continuous shaking. Subsequent synthesis was conducted in the same manner as the synthesis of

Dimer N-methylated peptides (M1-3)
The peptide was manually synthesized on Sieber amide resin (0.46 mmol/g). Resin (40 mg, 18.4 µmol) was swelled with DMF in a 2 mL fritted syringe with continuous shaking. Fmoc deprotection was performed by shaking the resin with 20% piperidine/DMF for 3 min. After washing the resin with DMF, the residual Fmoc group was removed by shaking the resin with 20% piperidine/DMF for 12 min, and the resin was washed with DMF three times. Fmoc protected N-methylamino acids (Fmoc-NMeLeu-OH or Fmoc-NMePhe-OH) (8 equivalent), HATU (8 equivalent), and DIPEA (16 equivalent) were dissolved in 736 µL of DMF, and the solution was added to the resin. The resin was incubated for 3 h at room temperature with continuous shaking. After the reaction, the resin was washed with DMF three times. After removal of the Fmoc group, N-terminal residue was coupled for 3 h using Fmoc-protected amino acids (Fmoc-Leu-OH or Fmoc-Phe-OH) (8 equivalent), HATU (8 equivalent), and DIPEA (16 equivalent) in 736 µL of DMF. After the reaction, the resin was washed with DMF three times. Subsequent synthesis was the same as the synthesis of D1-3. Purified peptides were dissolved in DMSO, yielding 10 mM peptides solution as DMSO stock. The concentration was determined based on the weight of the peptide. Purified peptides were analyzed by ESI-MS and UPLC.

D9.16-amide)
The hexapeptides were manually synthesized on 2-chlorotriryl chloride resin (1.6 mmol/g). For example, CP1 (cyclo[Tyr-Leu-D-Leu-Leu-Leu-D-Pro]) was synthesized as follows. Resin (20 mg, 32 µmol) was swelled with DCM in a 6 mL fritted syringe with continuous shaking. Fmoc-Leu-OH (2 equivalent) and DIPEA (4 equivalent) were dissolved in 640 µL DCM, and the solution was added to the resin. The resin was shaken overnight at room temperature. After the reaction, the resin was washed with DCM and 17:2:1 DCM:methanol:DIPEA three times each. The loading was quantified according to a previous report. 3 The resin was applied to further peptide synthesis. Fmoc deprotection was performed by shaking the resin with 20% piperidine/DMF for 3 min. After washing the resin with DMF, the residual Fmoc group was removed by shaking the resin with 20% piperidine/DMF for 12 min, and the resin was washed with DMF three times. The coupling reaction was performed by shaking the resin with Fmoc-protected amino acid (4 equivalent), HATU (4 equivalent), and DIPEA (8 equivalent) in DMF (0.1 M with respect to Fmoc-protected amino acid) for 1-1.5 h. After the reaction, the resin was washed with DMF three times. Above deprotection and coupling reactions were repeated until removal of the Fmoc group of N-terminal Leu-5. The resin was washed with DMF and DCM three times each. The linear precursor peptide was cleaved from the resin by shaking the resin with 20% 2,2,2-trifluoroethanol/DCM for 30 min twice. The filtrate was collected and evaporated. The peptide was dissolved in 30% acetonitrile/water and purified by HPLC. The purified peptide was lyophilized. For CP2-6, this purification step was omitted. The lyophilized peptide, PyAOP (1.5 equivalent), HOAt (1.5 equivalent), and DIPEA (4.5 equivalent) were dissolved to DMF (2 mM with respect to the peptide) and stirred overnight at room temperature to cyclize the peptide. After the reaction, the solvent was evaporated, and 50% TFA/45% DCM/2.5% TIPS/2.5% water was added and stirred 2 h at room temperature to perform the side chain deprotection. After deprotection, the solvent was evaporated. The peptide was dissolved to 30% acetonitrile/water, purified by HPLC, and lyophilized. After lyophilization, the purified peptide was dissolved in DMSO, yielding 10 mM CP1 solution based on the UV absorbance at 280 nm as DMSO sock. Purified CP1 was analyzed by ESI-MS and UPLC.
The 8-mer and 9-mer peptides were synthesized in 100 µmol scale using an automated peptide synthesizer (Syro I, Biotage). The C-terminal amino acid was first loaded on 2-chlorotriryl chloride resin with the same procedure with that used for CP1. The following peptides synthesis was conducted on Syro I using COMU as a coupling reagent. The synthesized linear peptides were cleaved from resin using 20% 1,1,1,3,3,3-hexafluoro-2-propanol in DCM. The peptides were cyclized using the same procedure for CP1 without a prior purification. The cyclized peptides were purified by HPLC and lyophilized. The purified peptides were analyzed by UPLC-MS. The 6-mer depsipeptides were manually synthesized on 2-chlorotriryl chloride resin (1.6 mmol/g). The ester bond was located at N-terminus in the linear precursor peptide because the repeated piperidine treatment may cause racemization and/or hydrolysis. For example, when it comes to cyclo Leu-Leu-Leu-OH was synthesized as the linear precursor peptide. The coupling of Fmoc-protected amino acids to ahydroxy acid was performed according to a previous report. 2 The peptide synthesis method was the same as that for the synthesis of CP1. Coupling of α-hydroxy acids was performed by shaking the resin with α-hydroxy acid (4 equivalent), HATU (4 equivalent), and DIPEA (8 equivalent) in DMF (0.2 M with respect to hydroxy acid) for 2 h at room temperature.
After the reaction, the resin was washed with DMF and THF three times each. The resin was shaken with Fmoc-protected amino acid (4 equivalent), DIC (4 equivalent), and DMAP (0.1 equivalent) in THF (0.1 M with respect to Fmoc-protected amino acid) for 2 h at room temperature. After the reaction, the resin was washed with THF and DMF three times each.
Subsequent synthetic procedures were the same as those for the synthesis of CP1. Purified peptides were analyzed by ESI-The 8-mer and 9-mer depsipeptides were synthesized in 100 mmol scale using an automated peptide synthesizer (Syro I, Biotage). The C-terminal amino acid was first loaded on 2-chlorotriryl chloride resin with the same procedure with that for CP1. The following peptides synthesis was conducted on Syro I using COMU as a coupling reagent. The coupling reactions of a hydroxy acid and the following N-terminal amino acid were conducted manually with the same procedure used for DP1. The synthesized linear peptides were cleaved from resin using 20% 1,1,1,3,3,3-hexafluoro-2propanol in DCM. The cleaved peptides were dissolved purified by HPLC. The purified peptides were lyophilized. The peptides were cyclized using the same procedure for CP1. The cyclized peptides were purified by HPLC and lyophilized.

Cyclic N-methylated peptides
The synthesized linear peptides were cleaved from resin using 20% 1,1,1,3,3,3-hexafluoro-2-propanol in DCM. The peptides were cyclized using the same procedure for CP1 without a prior purification. The cyclized peptides were purified by HPLC and lyophilized. The purified peptides were analyzed by UPLC-MS.
Compound 1, the chloroalkane carboxylic acid, was synthesized according to the previous report.

Purity check of synthesized compounds
The purities of the products were analyzed on UPLC monitored at 220 nm. UPLC analysis was performed using a linear gradient of solvent A (water containing 0.1% TFA) and solvent B (acetonitrile containing 0.1% TFA). The blue line denotes the percentage of solvent B.

Supplementary Figure 1. 1 H-NMR spectrum of DP2 for conformational analysis
The 1 H-NMR spectrum of DP2 was recorded in CDCl3. Light blue, orange, and green letters denote peaks derived from HN, HCα, HCβ, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 2. COSY spectrum of DP2 for conformational analysis
The COSY-NMR spectrum of DP2 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with red dashed lines.

Supplementary Figure 3. TOCSY spectrum of DP2 for conformational analysis
The TOCSY-NMR spectrum of DP2 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with red dashed lines.

Supplementary Figure 4. HSQC-NMR spectrum of DP2
The HSQC-NMR spectrum of DP2 was recorded in CDCl3. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 5. HMBC-NMR spectrum of DP2
The HMBC-NMR spectrum of DP2 was recorded in CDCl3. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 10. 1 H-NMR spectrum of CP1 for conformational determination
The 1 H-NMR spectrum of CP1 was recorded in CDCl3. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow, and pink letters denote peaks derived from HN, HCα, HCβ, HCd, HCg, and Haryl, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 11. COSY spectrum of CP1 for conformational determination
The COSY-NMR spectrum of CP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 12. TOCSY spectrum of CP1 for conformational determination
The TOCSY-NMR spectrum of CP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 13. HSQC spectrum of CP1 for conformational determination
The HSQC-NMR spectrum of CP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 14. HMBC spectrum of CP1 for conformational determination
The HMBC-NMR spectrum of CP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 15. ROESY spectrum of CP1 for conformational determination
The ROESY-NMR spectrum of CP1 was recorded in CDCl3. Correlation peaks indicating the proximities of protons are indicated with the names of the proximal two protons. The correlation peaks are summarized in a Supplementary Table.

Supplementary Figure 16. 1 H-NMR of DP1 for conformational determination
The 1 H-NMR spectrum of DP1 was recorded in CDCl3. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow, and pink letters denote peaks derived from HN, HCα, HCβ, HCg, HCd, and Haryl, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs. HCβ, HCd, and HCg were also assigned, but not all the assignment was shown because of their severe overlaps.

Supplementary Figure 17. COSY spectrum of DP1 for conformational determination
The COSY-NMR spectrum of DP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 18. TOCSY spectrum of DP1 for conformational determination
The TOCSY-NMR spectrum of DP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 19. HSQC spectrum of DP1 for conformational determination
The HSQC-NMR spectrum of DP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 20. HMBC spectrum of DP1 for conformational determination
The

Supplementary Figure 23. 1 H-NMR of MP1 for conformational determination
The 1 H-NMR spectrum of MP1 was recorded in CDCl3. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow, pink, and black letters denote peaks derived from HN, HCα, HCβ, HCg, HCd, Haryl, and N-methyl protons, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs. HCβ, HCd, and HCg, were also assigned, but not all the assignment was shown because of their severe overlaps.

Supplementary Figure 24. COSY spectrum of MP1 for conformational determination
The COSY-NMR spectrum of MP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 25. TOCSY spectrum of MP1 for conformational determination
The TOCSY-NMR spectrum of MP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 26. HSQC spectrum of MP1 for conformational determination
The HSQC-NMR spectrum of MP1 was recorded in CDCl3. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 27. HMBC spectrum of MP1 for conformational determination
The

Supplementary Figure 30. 1 H-NMR of CP1 in 50% DMSO/H2O
The 1 H-NMR spectrum of CP1 was recorded in 50% DMSO/H2O. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow, and pink letters denote peaks derived from HN, HCα, HCβ, HCd, HCg, and Haryl, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 31. COSY-NMR of CP1 in 50% DMSO/D2O
The COSY-NMR spectrum of CP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 32. TOCSY spectrum of CP1 in 50% DMSO/H2O
The TOCSY-NMR spectrum of CP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 33. HSQC spectrum of CP1 in 50% DMSO/D2O
The HSQC-NMR spectrum of CP1 was recorded in 50% DMSO/D2O. Correlation peaks that support the assignment of

Supplementary Figure 34. HMBC spectrum of CP1 in 50% DMSO/D2O
The HMBC-NMR spectrum of CP1 was recorded in 50% DMSO/D2O. The magnified spectrum with the correlation area of carbonyl carbon and HCα was shown. Correlation peaks that support the assignment of 1 H-NMR are shown with pink dashed lines.

Supplementary Figure 36. 1 H-NMR of DP1 in 50% DMSO/H2O
The 1 H-NMR spectrum of DP1 was recorded in 50% DMSO/H2O. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow and pink letters denote peaks derived from HN, HCα, HCβ, HCd, HCg, and Haryl, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 37. COSY-NMR of DP1 in 50% DMSO/H2O
The COSY-NMR spectrum of DP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 38. TOCSY spectrum of DP1 in 50% DMSO/H2O
The TOCSY-NMR spectrum of DP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 39. HSQC spectrum of DP1 in 50% DMSO/D2O
The HSQC-NMR spectrum of DP1 was recorded in 50% DMSO/D2O. Correlation peaks that support the assignment of

Supplementary Figure 40. HMBC spectrum of DP1 in 50% DMSO/H2O
The HMBC-NMR spectrum of DP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 42. 1 H-NMR of MP1 in 50% DMSO/H2O
The 1 H-NMR spectrum of MP1 was recorded in 50% DMSO/H2O. For the assignment of each peak, COSY, TOCSY, HSQC, HMBC, and ROESY spectra were recorded. Light blue, orange, green, purple, dark yellow, and pink letters denote peaks derived from HN, HCα, HCβ, HCd, HCg, and Haryl, respectively. The one-letter residue code and the number above a peak indicate the residue number to which the proton belongs.

Supplementary Figure 43. COSY-NMR of MP1 in 50% DMSO/D2O
The COSY-NMR spectrum of MP1 was recorded in 50% DMSO/D2O. Correlation peaks that support the assignment of

Supplementary Figure 44. TOCSY spectrum of MP1 in 50% DMSO/H2O
The TOCSY-NMR spectrum of MP1 was recorded in 50% DMSO/H2O. Correlation peaks that support the assignment of

Supplementary Figure 45. HSQC spectrum of MP1 in 50% DMSO/D2O
The HSQC-NMR spectrum of MP1 was recorded in 50% DMSO/D2O. Correlation peaks that support the assignment of

Supplementary Figure 46. HMBC spectrum of MP1 in 50% DMSO/D2O
The HMBC-NMR spectrum of MP1 was recorded in 50% DMSO/D2O. Correlation peaks that support the assignment of

Supplementary Figure 51. 1 H-NMR spectrum of HO-Tyr(tBu)-OH
The 1 H-NMR spectrum of HO-Tyr(tBu)-OH was recorded in CDCl3 at 298 K using an ECS-400 (JEOL) with a magnetic field of 400 MHz. Residual internal CHCl3 (δ 7.26) was used as the standard.

Supplementary
The Caco-2 assay was conducted under the same conditions as described in the Methods section other than that the incubation time was changed from 3 h to 2 h.